Hepatocyte transplantation has an immense potential for gene therapy and for treating acute liver failure. Because hepatocytes possess the cellular machinery to express a variety of genes, their use for ex-vivo gene therapy is particularly attractive. Similarly, hepatocyte transplantation should provide novel therapies for acute liver failure, which currently results in unacceptable mortalities of greater than 80%. Although orthotopic liver transplantation can be employed in acute liver failure, this is a formidable and irreversible procedure. In contrast, advantages of hepatocyte transplantation include its technical simplicity, the possibility of transplanting cells prepared from a single liver into multiple recipients and the potential to cryopreserve and transplant cells at a short notice. However, despite a great urgency in applying hepatocyte transplantation in humans, a better understanding of the biology of transplanted hepatocytes is required for an optimal usage. We hypothesize that the success of hepatocyte transplantation requires a) identification of permissive conditions for hepatocyte survival and function, b) identification of methods to increase the mass of transplanted hepatocytes, and c) optimization of the time and dose of cells required for therapeutic applications. To begin addressing these critical issues, we developed novel systems with genetically marked hepatocytes to unequivocally distinguish between transplanted and host cells. Also, we demonstrated that transplanted hepatocytes survive and function most optimally in the liver. We now propose to examine mechanisms regulating proliferation and fate of transplanted hepatocytes in liver. We will examine the proliferative capacity of transplanted hepatocytes and determine whether use of liver regenerative stimuli could augment the mass of transplanted hepatocytes. As transplantation of hepatocytes into the liver may be limited by the capacity of the hepatic vascular bed, we will determine the safety of hepatocyte transplantation. Using insights derived from these studies, we will develop strategies for massive reconstitution of the host liver. These strategies will be tested in powerful animal models of genetic metabolic disease. Also, animal models of acute liver failure will be used to define the timing, dose and value of hepatocyte transplantation, a well as simultaneous administration of hepatic growth factors. Finally, hepatocyte precursor cells will be used to determine their value compared with primary hepatocytes. Completion of these proposed studies will greatly advance our fundamental knowledge of hepatocyte transplantation and help move this technology from the laboratory to the bedside.